The liver, an organ which is present in vertebrates and other animals, plays a major role in the metabolism and has a number of functions in the body, including glycogen storage, decomposition of red blood cells, synthesis of plasma proteins, and detoxification. The liver also is the largest gland in the human body. It lies below the diaphragm in the thoracic region of the abdomen. It produces bile, an alkaline compound which aids in digestion, via the emulsification of lipids. It also performs and regulates a wide variety of high-volume biochemical reactions requiring specialized tissues.
Hepatocytes make up 70 to 80% of the cytoplasmic mass of the liver. Hepatocytes are involved in protein synthesis, protein storage and transformation of carbohydrates, synthesis of cholesterol, bile salts and phospholipids, and detoxification, modification and excretion of exogenous and endogenous substances. The hepatocyte also initiates the formation and secretion of bile.
There is a wide number of known liver diseases, such as:                Hepatitis: inflammation of the liver, caused mainly by various viruses but also by certain poisons, autoimmunity or hereditary conditions;        Cirrhosis: the formation of fibrous tissue in the liver, replacing dead liver cells. The death of the liver cells can for example be caused by viral hepatitis, alcoholism or contact with other liver-toxic chemicals;        Haemochromatosis: a hereditary disease causing the accumulation of iron in the body, eventually leading to liver damage;        Cancer of the liver: primary hepatocellular carcinoma (HCC) or cholangiocarcinoma and metastatic cancers, usually from other parts of the gastrointestinal tract;        Wilson's disease: a hereditary disease which causes the body to retain copper;        Primary sclerosing cholangitis: an inflammatory disease of the bile duct, autoimmune in nature;        Primary biliary cirrhosis: autoimmune disease of small bile ducts;        Budd-Chiari syndrome: obstruction of the hepatic vein;        Gilbert's syndrome: a genetic disorder of bilirubin metabolism, found in about 5% of the population;        Glycogen storage disease type II: the build-up of glycogen causes progressive muscle weakness (myopathy) throughout the body and affects various body tissues, particularly in the heart, skeletal muscles, liver and nervous system.        
There are also many pediatric liver diseases, such as biliary atresia, alpha-1-antitrypsin deficiency, alagille syndrome, and progressive familial intrahepatic cholestasis; as well as metabolic diseases.
Furthermore, several pathogens and parasites, especially of tropical diseases, have a liver stage during their life cycle. For instance malaria, is one of the most common infectious diseases and an enormous public-health problem. Malaria is caused by protozoan parasites of the genus Plasmodium. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species (Plasmodium ovale, Plasmodium malariae, and sometimes Plasmodium knowlesi) can also infect humans. This group of human-pathogenic Plasmodium species is usually referred to as malaria parasites. Malaria parasites are transmitted by female Anopheles mosquitoes. Malaria in humans develops via two phases: an exoerythrocytic (hepatic phase or “liver stage”) and an erythrocytic phase. When an infected mosquito pierces a person's skin to take a blood meal, sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver. Within about 30 minutes of being introduced into the human host, they infect hepatocytes, multiplying asexually and asymptomatically for a period of 6-15 days. Once in the liver these organisms differentiate to yield thousands of merozoites which, following rupture of their host cells, escape into the blood and infect red blood cells, thus beginning the erythrocytic stage of the life cycle. The parasite escapes from the liver undetected by wrapping itself in the cell membrane of the infected host liver cell. The parasite is relatively protected from attack by the body's immune system because for most of its human life cycle it resides within the liver and blood cells and is relatively invisible to immune surveillance. There is increasing interest in developing drugs that specifically address the liver specific stages of malaria parasites (e.g. primaquine, gyrase inhibitors like levofloxacin, doxorubicin) in order to obviate the development of blood stages.
Another example is schistosomiasis or bilharziosis, which is a parasitic disease caused by several species of flatworm. Although it has a low mortality rate, schistosomiasis can be very debilitating. It is an often chronic illness that results from infection of the blood with a parasitic flatworm (schistosome). It causes debilitation and causes liver and intestinal damage. It is most commonly found in Asia, Africa, and South America, especially in areas with water that is contaminated with fresh water snails, which contain the parasite.
Hepatitis B virus (HBV), the cause for hepatitis B, is the prototype of a family of small, enveloped DNA viruses of mammals and birds (1). The HBV envelope encloses three proteins termed L-(large), M-(middle) and S-(small) (see FIG. 1). They share the C-terminal S-domain with four transmembrane regions. The M- and L-protein carry additional N-terminal extensions of 55 and, genotype-dependent, 107 or 118 aa (preS2- and preS1). In virions the stoichiometric ratio of L, M and S is about 1:1:4, while the more abundantly secreted non-infectious subviral particles (SVPs) contain almost exclusively S- and only traces of L-protein (2). During synthesis, the preS1-domain of L is myristoylated and at some point of the HBV life cycle translocated through the ER-derived membrane. This modification is essential for infectivity (3,4). A pronounced feature of HBV is its liver tropism, i.e. the liver is the tissue which specifically supports the growth of HBV.
Ideally, drug targeting should fulfil the following criteria: 1) exclusive transfer of the drug to the required site of action; 2) a minimum of effects for the remaining organism; 3) use of a pharmacologically inactive vector.
In order to carry a drug i.e. a therapeutic active agent to a specific tissue, different strategies are pursued. These are for example the use of prodrugs, from which the pharmacologically active part is released in the target tissue by tissue-specific enzymes. A further possibility is to couple effective, non-tissue-specific drugs to tissue-specific, but pharmacologically inert carrier systems like receptor-affine peptides or colloidal particles.
Various drug carriers have been used to enhance liver targeting of drugs. A straightforward approach is based on the active phagocytosis of the reticuloendothelial system in the liver by delivering drugs in particular carriers, such as liposomes and microspheres. For example, it has been shown that following i.v. (intravenous) injection, particulate carriers incorporating a drug are mainly captured by the reticuloendothelial system in the liver, resulting in drug targeting of the liver (5). On the other hand, liver targeting of drugs with positively charged, water-soluble polymers is based on free extravasation of most water-soluble substances from the vascular system of the liver as well as on negative charges on the liver cell surface (6). Thus, polymers have been used as the carrier to allow drugs to target to the liver based on such anatomical and biochemical characteristics of the liver. More specific drug targeting of the liver has been attempted by using asialoglycoprotein receptors of liver cells. The asialoglycoprotein receptor (galactose receptor) is present on hepatocytes with high density. In addition, once a ligand binds to the galactose receptor, the ligand-receptor complex is internalized which allows the cellular uptake of galactosylated ligands (7). Furthermore delivery approaches using nanoparticles have been performed by e.g. amphilic polymers and viral vectors (8). Also delivery of drugs and genetic material has been conducted via the use of bio-nanocapsules (BNCs). BNCs are described as “nano-scaled capsules consisting of proteins produced by biotechnological techniques” and can be used as delivery systems for organ specific drug delivery (9).
U.S. Pat. No. 7,001,760 B2 disclose recombinant vectors derived from hepatitis B virus (HBV), which can be used for gene therapy, such as the delivery of therapeutic genes to liver cells and the expression of heterologous genes in liver cells.
WO2009/092612, whose content is incorporated herewith by reference in its entirety, describes hydrophobic modified preS-derived peptides of HBV and their use as vehicles for the specific delivery of compounds to the liver. In this document, the hydrophobic modified preS-derived peptides of HBV may be coupled with a diagnostic or therapeutic active agent via an optional anchor group (A) which is preferably “C-terminal” of the hydrophobic modified preS-derived peptide.
The present invention provides hydrophobic modified peptides conjugated to one ore more compounds, preferably being drug(s), which compounds are coupled to the N-terminal amino acid sequence of the peptide represented by X, making it possible to create shorter peptides while still maintaining liver specificity. Surprisingly it has become possible to couple hydrophobic moieties to the peptides without abrogating liver tropism. The coupling compounds like drugs to the N-terminal site allows better delivery of active compounds across the cell membrane and also allows to direct drugs, which are active on the cellular membrane, directly to the site of action. Furthermore, the coupling of a drug to the N-terminal region of the peptide contributes to the stability of this active substance. In FIG. 3, the molecular mechanism of the binding of a hydrophobic modified peptide to the surface of a hepatocyte is schematically illustrated.